Novel liposome complexes for increased systemic delivery

ABSTRACT

Highly efficient cationic liposomes have been developed as an improved delivery system for biologically-active reagents. A novel structure, the sandwich liposome, is formed and comprises one or more biologically active agents internalized between two bilomellar liposomes. This structure protects the incoming agent and accounts for the high efficiency of in vivo delivery and for the broad tissue distribution of the sandwich liposome complexes.  
     These novel liposomes are also highly efficient carriers of nucleic acids. By using extruded DOTAP:cholesterol liposomes to form complexes with DNA encoding specific proteins, expression has been improved dramatically. Highest expression was achieved in the lung, while increased expression was detected in several organs and tissues.

FIELD OF THE INVENTION

[0001] The present invention is directed to a liposomal preparationwhich is based on a composition of specific lipids which form liposomes.It is also an object of the present invention to provide a method forpreparing a liposomal composition carrying a biologically active agent.The liposomal delivery system of the present invention is used as highlyefficient transfer and therapy methods.

BACKGROUND OF THE INVENTION

[0002] Lipidic particles may be complexed with virtually any biologicalmaterial. This capability allows these complexes to be useful asdelivery systems for proteins, therapeutic agents, chemotherapeuticagents and nucleic acids. Although lipidic complexes have been used fora myriad of drug therapies, one area where these delivery systems haveshown promising results is in gene therapy. For gene therapy to besuccessful efficient and safe transfer of genes or biologically activereagent to a target cell is required. Hence the need for improveddelivery systems, in both conventional and gene-based therapies isalways at the forefront.

[0003] Lipidic particles have been shown to be efficient vehicles formany in vitro and in vivo applications. Lipidic particles complexed withDNA have been used in vitro (Felgner et al. (1987); Gao et al. (1991))and in vivo (Nabel et al. (1990); Wang et al. (1987); Zhu et al. (1993);Soriano et al. (1983)) for the expression of a given gene through theuse of plasmid vectors.

[0004] Formation of complexes of DNA with cationic lipidic particles hasrecently been the focus of research of many laboratories. Improvedformulations of cationic lipids have greatly increased the efficiency ofDNA delivery to cells in tissue culture (Felgner et al. (1987)). Incontrast, intravenous DNA delivery in animals using cationic liposomeshas been less efficient (Zhu et al. (1993); Philip et al. (1993);Solodin et al. (1995); Liu et al. (1995); Thierry et al. (1995);Tsukamoto et al. (1995); Aksentijevich et al. (1996)) limiting thetherapeutic application of nonviral vectors to gene therapy. Improvedliposome formulations for in vivo delivery is a valuable alternative togene therapy using viral vectors and avoids several problems associatedwith viral delivery. Although efforts to synthesize new cationic lipidsled to the discovery of more efficient transfection agents, theirefficiency measured in tissue culture does not correlate with ability todeliver DNA after systemic administration in animals (Solodin et al.(1995)). Functional properties defined using in vitro experiments do notassess stability of the complexes in plasma or their pharmacokineticsand biodistribution, all of which are essential for in vivo activity(Felgner et al. (1994)). Colloidal properties of the complexes inaddition to the physicochemical properties of their component lipids maydetermine these parameters.

[0005] The liposome provides an alternative to viral delivery systems ingene therapy which may involve the transfer of normal, functionalgenetic material into cells to correct an abnormality due to a defectiveor deficient gene product. Typically, the genetic material to betransferred should at least contain the gene to be transferred togetherwith a promoter to control the expression of the new gene.

[0006] Methods for viral DNA delivery systems suffer from many inherentproblems including immune responses, inability to deliver viral DNAvectors repeatedly, difficulty in generating high viral titers, and thepossibility of infectious virus. Non-viral delivery methods provide analternative system that is devoid of these problems. However, until now,low efficiency of DNA delivery by liposomes has limited the therapeuticapplication of this technology for gene therapy. The liposomes of thepresent invention have increased systemic DNA delivery and geneexpression up to 150-fold over that previously reported.

[0007] Therefore, an object of the present invention is the synthesis ofa highly efficacious liposome structure capable of deliveringbiologically active agents into a subject.

[0008] Another object of the present invention is the use of thesestable liposomes as carriers of nucleic acids for delivery andexpression of the nucleic acid product at a target site within ananimal.

[0009] Yet another object of the present invention is the use of thesestable liposomes as carriers of nucleic acids for delivery andexpression of the nucleic acid product systemically to a patient.

[0010] A further object of the present invention is the production ofkits containing the stable liposomes of the present invention, capableof carrying any nucleic acid of interest.

[0011] Another object of the present invention is to use liposomescarrying specific reagents for human gene therapy in treatment ofdisease.

[0012] Yet a further object of the present invention relates toproviding a method for long-term expression of a gene product from anon-integrated nucleic acid in a patient.

SUMMARY OF THE INVENTION

[0013] The present invention relates to a novel liposome structurecapable of carrying biologically-active reagents. These liposomestructures are highly efficient vehicles for delivery of biologicallyactive agents to target locations in a patient as well as providesystemic reagent delivery. The liposome complexes of the presentinvention are small in size and have a net ± charge (“ρ”) of about 2.

[0014] The present invention further relates to a method of preparingthese novel liposomes comprising the steps of heating, sonicating, andextrusion of the liposome structures. The method of preparation of thepresent invention produces complexes of appropriate and uniform size,which are structurally stable and produce maximal extrusion. Liposomesprepared by this method are also encompassed by the present invention.

[0015] The present invention further relates to a novel liposomestructure capable of carrying nucleic acids. The present invention alsorelates to an improved liposome formulation comprising DOTAP(1,2-bis(oleoyloxy)-3-(trimethylammonio)-propane) and cholesterol(“Chol”) and a nucleic acid which produces exceptionally high geneexpression and protein production in vivo. These formulations areextremely stable, homogeneous in size, and can complex nucleic acidsover a wide range of nucleic acid:liposome ratios. The present inventiondemonstrates up to 150-fold greater gene expression following in vivosystemic delivery in animals as compared to formulations previouslydescribed in the literature.

[0016] The present invention also relates to liposomes carryingnon-immunogenic targeting ligands and stealth lipids. These ligandsfacilitate the targeted delivery of the liposomes to a particular tissueor site in the body.

[0017] The present invention relates to kits containing the presentliposome structure capable of carrying a reagent within it. One such kitmay comprise the liposome structures ready for the user to add thebiological reagent of interest. A kit may further comprise a liposomepreparation and one or more specific biologically-active reagents foraddition to the liposome structure. Another kit of the present inventioncomprises a set of liposome structures, each containing a specific,biologically-active reagent, which when administered together orsequentially, are particularly suited for the treatment of a particulardisease or condition.

[0018] The present invention provides a therapeutic method of treatingdiseases, ailments and conditions based upon a liposome-facilitateddelivery of biologically active agents. For example, the presentinvention provides a pharmaceutical liposomal formulation for thedelivery of nucleic acids using systemic administration to providelong-term expression of a given nucleic acid. In addition, the presentinvention encompasses in vitro cell transfection followed by tissuetransplantation such that the transfected cells may be incorporated intransplanted tissue. This method is referred to as in vitro/ex vivotransfer. Other biologically-active agents may be encapsulated in theliposomes of the present invention for in vitro/ex vivo methods so longas a ± charge of positive 2 is maintained.

[0019] The present invention further provides an effective vaccinevehicle capable of effective delivery, boosting antigen-immune responseand lowering unwanted extraneous immune response, presently experiencedwith adjuvants.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1A. Phase Diagram for DOTAP:Chol-DNA liposome complexes.Liposomes were prepared (see Example 1) and complexed with DNA (seeExample 2) at various concentrations of DNA and liposomes in a finalvolume of 200 μl. The absorbance at 400 nm was determined for 1:20dilutions of each DNA:liposome complex and plotted. An absorbance of 2.0indicates precipitation of the complexes. White boxes indicate datapoints not determined.

[0021]FIG. 1B. Production of Chloramphenicol Acetyl Transferase (“CAT”)in the mouse lung following systemic delivery using a variety ofDNA:liposome formulations. Each mouse was injected with 82.5 μg plasmidDNA:5 mM liposome (5 mM cationic lipid+5 mM neutral lipid), the optimalDNA:lipid ratio for the DDAB (Dimethyldioctadecylammonium Bromide)formulations. CAT expression was normalized to protein concentration inthe tissue extracts and is presented as the mean±Standard Error of theMean (“SEM”) of duplicate determinations.

[0022]FIG. 2A. CAT production in vivo following systemic delivery ofDOTAP:Chol-DNA complexes. Dose response in the lung using variousDOTAP:Chol-DNA:liposome ratios. The concentrations of DOTAP:Chol variedfrom 0.5 mM to 6 mM complexed with 50 to 175 μg DNA per tail veininjection. (All mM liposome concentrations refer to the cationic lipidor the neutral lipid; therefore, 6 mM DOTAP:Chol=6 mM DOTAP+6 mM Chol.)5 mM DOTAP:Chol was complexed to 50, 100, 125, 150, and 175 μg DNA. At100 μg DNA, DOTAP:Chol was complexed at 5.5 mM, 5 mM, 4.75 mM, 4.5 mM, 4mM, and 3 mM. At 50 μg DNA, DOTAP:Chol was complexed at 6 mM, 5 mM, 4mM, 3 mM, 2 mM, 1 mM, and 0.5 mM. The data points shown here areaverages of CAT production from 40 mouse lungs. Each data point is atleast a duplicate determination, and some data points were averages of 4assays with standard errors varying from 1% to 18% of the mean. Whiteboxes indicate data points not determined.

[0023]FIG. 2B. CAT production in various tissues using 3 mM DOTAP:Cholcomplexed with 100 μg DNA per tail vein injection. CAT expression wasnormalized to protein concentration in the tissue extracts and ispresented as the mean±SEM.

[0024]FIG. 2C. Dose response in the heart using various DNA:liposomeratios showing total CAT production. The concentrations of DOTAP:Cholvaried from 3 mM to 5.5 mM complexed with 100 μg DNA per tail veininjection.

[0025]FIG. 2D. Comparison of CAT production levels in the lungs of miceexsanguinated prior to organ harvest and mice that were not bled. Inaddition, whole blood was assayed. CAT activity in the lung is due togene expression in tissue rather than in blood. CAT expression wasnormalized to protein concentration in the tissue extracts and ispresented as the mean±SEM.

[0026]FIG. 2E. Comparison of CAT production in the lungs ofexsanguinated mice using different DOTAP formulations. All of thefollowing formulations shown used 4 mM DOTAP: DOTAP:Chol (50:50), DOTAP,DOTAP:Chol:DOPS (50:45:5), DOTAP:DOPE (50:50), and DOTAP:DOPC (50:50).“DOPS” is dioleoyl (18:1) phosphatidyl serine “DOPC” is oleoyl (18:1)phosphatidyl choline (9-cis) octadecanoic acid. All formulations werecomplexed to 100 μg of DNA in a 200 μl final volume. CAT expression wasnormalized to protein concentration in the tissue extracts and ispresented as the mean±SEM.

[0027]FIG. 3. Cryoelectron micrographs of liposomes and DNA:liposomecomplexes. (A) 5 mM DOTAP:Chol liposomes. A thin film was prepared bydipping and withdrawing a 700 mesh copper grid (3 mm diameter, 3 to 4 μmthick) in the DOTAP:Chol suspension. After blotting away excess liquid,the thin films that form between the bars of the grid were vitrified inmelting ethane. After cryotransfer, the specimen was observed at −170°C. in a Philips CM 12 microscope at low dose, 120 kV (Frederik et al.(1991)). Note the continuity between concentric bilayers in some of thevesicles, giving the shape of a vase with an orifice. (B) 5 mMDOTAP:Chol liposomes mixed with 150 μg DNA/200 μl. These structures arereferred to as DNA-sandwich liposomes. A thin vitrified specimen wasprepared from this DNA:liposome suspension and observed at −170° C. (asin A). Note the packing of DNA (electron opaque addition to the lipid)in the interior of the vesicles. Lipid-DNA interaction has apparentlyresulted in remodeling of the vesicles and changed the accessibility ofDNA. (C) Enlarged image of a DOTAP:Chol vase. (D) Enlarged image of DNApacked between two DOTAP:Chol vases. (E) 5 mM DOTAP:DOPE liposomes. (F)5 mM DOTAP:DOPE liposomes mixed with 150 μg of DNA in a 200 μl finalvolume.

[0028]FIG. 4. Proposed model showing cross-sections of DOTAP:Cholliposomes interacting with supercoiled plasmid DNA. The X indicatesfusion of lipid bilayers. The enlarged area shows proposed arrangementof DNA condensed between two 4 nm bilayers of DOTAP:Chol.

[0029]FIG. 5. Production of CAT in the mouse lung following systemicdelivery using a variety of DOTAP:Chol concentrations complexed to 100kg of DNA per tail vein injection. Concentrations of DOTAP:Chol usedvaried from 3.0 mM to 5.5 mM. The results showed slightly greater CATprotein production in the lung using 3.0 mM and 4.0 mM liposomescomplexed to 100 μg DNA per tail vein injection than the amount of CATproduced using 5.0 mM liposome complexed to 150 μg DNA. In addition, adose response was produced in vivo following injection of differentconcentrations of DOTAP:Chol used to make the DNA:liposome complexes.

[0030]FIG. 6. Production of CAT in the mouse lung following systemicdelivery using 5.0 mM DOTAP:Chol complexed to a variety of DNA amountsper tail vein injection. 5.0 mM DOTAP:Chol may be complexed to a widerange of plasmid DNA concentration without precipitation of theDNA:liposome complexes. Mice were injected with liposomes that contained50 μg, 100 μg, 125 μg, 150 μg and 175 μg of DNA plasmid per tail veininjection. It is not common for any liposome formulation to be highlytolerant over a wide range of DNA:liposome ratios; therefore, ourDOTAP:Chol liposomes are extremely unique in this regard. Furthermore,the present invention demonstrates a dose response in vivo usingdifferent amounts of DNA injected. At 150 μg DNA, the highest productionof CAT was produced in the lung, and that level of CAT production is upto 150-fold greater than any reported in the literature.

[0031]FIG. 7. CAT production in the mouse liver at 24 hours after tailvein injection using DNA:liposome complexes coated with succinylatedasialofetuin. Each mouse was injected into the tail vein with 4 mMDOTAP:Chol complexed to 100 μg of DNA in a 200 μl final volume with orwithout addition of succinylated asialofetuin after DNA:liposome mixing.CAT expression was normalized to protein concentration in the tissueextracts and is presented as the mean±SEM of duplicate determinations.

[0032]FIG. 8. Plot of the radially averaged intensity of DOTAP:Chol-DNAliposome complexes versus scattering wave-vector q. The DOTAP:Chol DNAliposome complexes show the x-ray diffraction maximum, 58.8A, thatconfirms the thickness of DNA+lipid determined by cryo-electronmicroscopy.

DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention relates to the discovery that liposomes ofa specific composition forming a stable structure are efficient carriersof biologically active agents. The liposome containing one or morebiologically active agents may then be administered into a mammalianhost to effectively deliver its contents to a target cell. The liposomesof the present invention are small and carry a net ± charge (referred toherein as “ρ”) of about 2 when complexed with a biologically activeagent. The liposomes are capable of carrying biologically active agents,such that the agents are completely sequestered. The liposomes comprisea cationic lipid, DOTAP and cholesterol or a cholesterol derivative.Preferably, at least one biologically active agent to be complexed withthe liposome is negatively charged. Additional biologically activeagents may be complexed with the liposome regardless of their charge, solong as ρ is maintained in the range of 1 to 3, preferably about 2. Theliposome-biologically active agent complex of the present inventionforms an invaginated structure referred to herein as a “sandwichliposome,” because the biologically active agents are sandwiched (andthus sequestered) between the lipid bilayers.

[0034] The present invention also provides a targeting means, such thatthe liposomes can be delivered to specific target sites. The targetingmeans comprises decorating the outside of the sandwich liposomecomplexes with one or more ligands specific for a particular target siteor sites.

[0035] “Biologically active agents” as the term is used herein refers tomolecules which affect a biological system. These include molecules suchas proteins, nucleic acids, therapeutic agents, vitamins and theirderivatives, viral fractions, lipopolysaccharides, bacterial fractionsand hormones. Other agents of particular interest are chemotherapeuticagents, which are used in the treatment and management of cancerpatients. Such molecules are generally characterized asantiproliferative agents, cytotoxic agents and immunosuppressive agentsand include molecules such as taxol, doxorubicin, daunorubicin,vinca-alkaloids, actinomycin and etoposide.

[0036] The term “nucleic acids” means any double strand or single stranddeoxyribonucleic acid (DNA) or ribonucleic acid (RNA) of variablelength. Nucleic acids include sense and anti-sense strands. Nucleic acidanalogs such as phosphorothioates, phosphoramidates, phosphonatesanalogs are also considered nucleic acids as that term is used herein.Nucleic acids also include chromosomes and chromosomal fragments.Potential genes include but are not limited to: immune system proteinsHLA-B7 and IL-2, cystic fibrosis transmembrane conductance regulator,Factor VIII, Factor IX, insulin and erythropoietin. (Felgner (1997)).

[0037] Antisense oligonucleotides may potentially be designed tospecifically target genes and consequently inhibit their expression. Inaddition this delivery system may be a suitable carrier for othergene-targeting oligonucleotides such as ribozymes, triple helix formingoligonucleotides or oligonucleotides exhibiting non-sequence specificbinding to particular proteins or other intracellular molecules. Forexample, the genes of interest may include retroviral or viral genes,drug resistance genes, oncogenes, genes involved in the inflammatoryresponse, cellular adhesion genes, hormone genes, abnormallyoverexpressed genes involved in gene regulation.

[0038] One embodiment of the present invention comprises encapsulating anucleic acid within a liposome and expressing a gene encoded on thenucleic acid within the target host cell, through the use of plasmidDNA. Conversely, the expression of a gene may be inhibited, for example,through the use of antisense oligonucleotides. Alternatively, achemotherapeutic agent may act as the biologically active agent and beencapsulated within a liposome, thereby sequestering its toxic effectsfrom non-targeted tissues.

[0039] The present invention may utilize more than one nucleic acid orbiologically active agent in the liposome of the present invention. Forexample, proteins such as DNA binding proteins can be added asadditional biologically active agents to DNA-sandwich liposomes tofacilitate a therapeutic effect. Another example includes sandwichliposomes carrying genes for anti-cancer treatment which are alsocarrying anti-cancer chemotherapeutic agents. This approach isespecially attractive when targeted liposomes are used to deliver bothgene therapy and chemotherapy specifically to cancer cells.

[0040] “Liposome” as the term is used herein refers to a closedstructure comprising of an outer lipid bi- or multi-layer membranesurrounding an internal aqueous space. In particular, the liposomes ofthe present invention form vase-like structures which invaginate theircontents between lipid bilayers (see FIG. 4). Liposomes can be used topackage any biologically active agent for delivery to cells. In oneexample, DNA can be packaged into liposomes even in the case of plasmidsor viral vectors of large size which may be maintained in a solubleform. Such invaginated liposome:DNA complexes are ideally suited fordirect application to in vivo systems. These liposomes entrap compoundsvarying in polarity and solubility in water and other solvents.

[0041] By “nucleic-acid-sandwich” liposomes is meant, the layeredcomposition comprising a structure having lipid bilayers with nucleicacid molecules inserted between and protected by the lipid layers.

[0042] One embodiment of the present invention relates to an improvedliposome formulation comprising a nucleic acid, DOTAP and cholesterolwhich produces exceptionally high gene expression and protein productionin vivo. In addition, these formulations are extremely stable,homogeneous in size, and can complex nucleic acids over a wide range ofnucleic acid:liposome ratios. This flexibility allows optimization ofthe complexes for delivery in vivo. Most tissues other than lung areextremely sensitive to the nucleic acid:liposome ratio. In addition, thestability of DOTAP:Chol liposomes at high concentrations of liposome andDNA allows for increased concentrations of DNA for delivery andexpression.

[0043] The present invention further relates to a method of preparingthese novel liposomes comprising the steps of heating, sonicating, andsequential extrusion of the lipids through filters of decreasing poresize, thereby resulting in the formation of small, stable liposomestructures. The method of preparation of the present invention producescomplexes of appropriate and uniform size, which are structurally stableand produce maximal extrusion.

[0044] Liposomes comprising DOTAP and at least one cholesterol and/orcholesterol-derivative, present in a molar ratio range of 2.0 mM-10 mMprovide an effective drug delivery system. More preferably, the molarratio of DOTAP to cholesterol is 1:1-3:1. The liposomal composition ofthe present invention has shown to be very stable in a biologicalenvironment.

[0045] Cholesterol derivatives may be readily substituted for thecholesterol element of the present liposome invention. Many cholesterolderivatives are known to the skilled artisan. Examples include but arenot limited to cholesteryl acetate and cholesteryl oleate.

[0046] Many DNA preparation protocols are available to the skilledartisan; any of which can be employed to prepare DNA for use in theliposomes of the present invention. Three DNA preparation protocols arepreferred, namely, alkaline lysis followed by PEG precipitation,anion-exchange chromatography (Qiagen), and a modified alkaline lysisprotocol (see Example 3). The modified alkaline lysis protocol is aparticularly preferred method to obtain high DNA yield, have low levelsof endotoxin, and achieve high levels of gene expression.

Transfer Therapy Methods

[0047] The liposomal composition of the present invention may beadministered into patients parenterally in order to achieve transfertherapy of one negatively-charged, biologically-active agent along withother biologically active agents. Moreover, this technique may be usedfor “ex vivo” transfer therapy where tissue or cells are removed frompatients, then treated and finally reimplanted in the patient (U.S. Pat.No. 5,399,346, describing the details of ex vivo human gene therapy isincorporated herein by reference). Alternatively, systemic therapy isalso effective in administering the liposome of the present invention.

[0048] Many diseases can be treated via the drug delivery system of thepresent invention. Diseases such as diabetes, atherosclerosis,chemotherapy-induced multi-drug resistance, and generally,immunological, neurological (Ho and Sapolsky (1997)) and viral diseases(Friedmann (1997)) can be treated using the present drug deliverysystem.

[0049] Non-limiting examples of gene therapy approaches for treatingcancer which can employ the delivery system of the present inventioninclude: antisense therapy (to block synthesis of proteins encoded bydeleterious genes), chemoprotection (to add proteins to normal cells toprotect them from chemotherapies), immunotherapy (to enhance the body'simmune defenses against cancer), pro-drug or suicide gene therapy (torender cancer cells highly sensitive to selected drugs), tumorsuppressor genes (to replace a lost or damaged cancer-blocking gene),antibody genes (to interfere with the activity of cancer-relatedproteins in tumor cells) and oncogene down-regulation (to shut off genesthat favor uncontrolled growth and spread of tumor cells) (Blaese(1997)).

[0050] The delivery system of the present invention is also useful forcorrecting the ion transport defect in cystic fibrosis patients byinserting the human CFTR (cystic fibrosis transmembrane conductanceregulator) gene. Oral administration such as nebulization may beparticularly suitable. In addition, the liposomes of the presentinvention can be used for the inhibition of tumor cells by administeringin tumor cells a molecule inhibiting tumorigenesis or a gene coding foran antisense polynucleotide directed to mRNA transcripts of angiogenicfactors. In addition, ribozymes may be encapsulated and enzymaticallyattack specific cellular contents.

[0051] The liposomes of the present invention containing the nucleicacid drug can be administered by intravenous, intramuscular,intraperitoneal, subcutaneous intra-lesional, oral or aerosol means.(Stribling et al. (1992)).

[0052] For aerosol administration, a patient receives one or more nasalor bronchial aerosol administrations of the liposome complex. Thedosages will vary based upon age, body composition and severity ofdisease or condition. (Caplen et al. (1995)).

[0053] Other routes of administration will be known to the skilledartisan and can be readily used to administer the liposomes of thepresent invention. Examples include but are not limited to mucosal,intra-uteral, intradermal and dermal.

[0054] A proposed daily dosage of active compound for the treatment ofhumans is 0.1 μg DNA/kg to 5.0 mg DNA/kg, which may be convenientlyadministered in 1-10 doses. The actual dosage amount administered can bedetermined by physical and physiological factors such as body weight,severity of condition, idiopathy of the patient and on the route ofadministration. With these considerations in mind, the dosage ofDNA-liposome complex for a particular subject and/or course of treatmentcan readily be determined.

[0055] The liposome of the invention may be formulated for parenteraladministration by bolus injection or continuous infusion. Formulationfor injection may be presented in unit dosage form in ampoules, or inmulti-dose containers with an added preservative. The compositions maytake such forms as suspension, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for reconstitution with a suitablevehicle, e.g. sterile pyrogen-free water, before use.

[0056] The liposomes according to the invention may be formulated foradministration in any convenient way. The invention therefore includeswithin its scope pharmaceutical compositions comprising at least oneliposomal compound formulated for use in human or veterinary medicine.Such compositions may be presented for use with physiologicallyacceptable carriers or excipients, optionally with supplementarymedicinal agents. Conventional carriers can also be used with thepresent invention.

[0057] For oral administration, the pharmaceutical composition may takethe form of, for example, tablets, capsules, powders, solutions, syrupsor suspensions prepared by conventional means with acceptableexcipients.

[0058] An improved method for preparing the liposomes of the presentinvention employs sonication, heating, and extrusion (see Example 1 fordetailed description). Generally, the method requires that the lipidcomponents be mixed in the appropriate concentrations, dissolved in anorganic solvent, such as chloroform or the like, evaporated into a thinfilm and lyophilized. The film is then rehydrated in an aqueous solutionand mixed for a period at 35°-60° C. Thereafter, the mixture issonicated and heated to a temperature between 40° C.-60° C. This mixtureis then sequentially extruded through filters of decreasing size.Sonicated liposomes are preferably extruded through filters ofdecreasing pore sizes, including 0.1 μm, by using sufficient heating.

[0059] Various nucleic acids may be added to these liposomes in a widerange of concentrations. Nucleic acids are preferably added to theliposomes at a concentration of 50-300 μg per dose. These concentrationsvary widely depending upon the ratio of DOTAP:Chol in the particularliposome preparation. For example, if a liposome having a molar ratio ofDOTAP:Chol of about 1:1 is used, then a preferred concentration ofnucleic acid is between 80-175 μg per dose.

[0060] Extruded DOTAP:Chol liposomes prepared by the method of thepresent invention were compared to non-extruded DOTAP:Chol liposomes(multilamellar vesicles, MLVs) prepared by the conventional method. (Liuet al. (1997)). The extruded liposomes form sandwich liposomes whereasthe non-extruded liposomes do not form such structures. Proteinexpression from nucleic acid: sandwich liposomes was approximately2-fold higher as compared to MLVs. Furthermore, MLVs containing 5 mM to10 mM DOTAP:Chol have increased toxicity when delivered systemically.

[0061] Production of chloramphenicol acetyl transferase (“CAT”) is themost widely used method to measure protein expression and is a wellrecognized model protein expression system. (Wheeler et al. (1996); Leeet al. (1996); Felgner (1996)). In one embodiment of the presentinvention the sandwich liposome complexes (i.e. FIG. 2A, D, E) produced100-fold greater amounts of CAT in the lung compared to CAT producedusing DOTIM:Chol SUVs. (Solodin et al. (1995)). Those skilled in the artwill readily recognize that any nucleic acid can be used in the presentinvention.

[0062] By using sandwich liposomes for systemic nucleic acid delivery, abroad biodistribution is produced (i.e. FIG. 2B) that is greater thanthat produced by any other cationic liposome formulation reported. (Zhuet al. (1993); Philip et al. (1993); Solodin et al. (1995); Liu et al.(1995); Thierry et al. (1995); Tsukamoto et al. (1995); Aksentijevich etal. (1996); Wheeler et al. (1996); Liu et al. (1997); Hong et al.(1997); Lee et al. (1996); Felgner (1996)).

Targeted Delivery

[0063] Because the biologically active agent is sequestered in thesandwich liposomes, targeted delivery is achieved by the addition ofpeptides and other ligands without compromising the ability of theseliposomes to bind and deliver large amounts of the agent. The ligandsare added to the liposomes in a simple and novel method. First, thelipids are mixed with the biologically active agent of interest. Thenligands are added directly to the sandwich-liposomes to decorate theirexterior surface. The stability and net positive charge of the liposomesallow ligands to be directly added to their exterior.

[0064] The sandwich liposome complexes of the present invention may beused to make effective artificial viruses. Because the outside of thesandwich liposome complexes is substantially free of biologically activeagents, targeting ligands may be placed on the outside after sandwichliposome formation, without compromising the effect of the targetingligand or the ability of the biologically active agents to be deliveredand expressed. This may enable delivery to specific organs and tissues.The size of the sandwich liposome complexes responsible for efficientdelivery, 200 to 450 nm (see also Table 1), is preferred for theaddition of targeting ligands. Our experiments demonstrate theusefulness of this approach (See Example 9, FIG. 7).

[0065] Many ligands may be employed for this targeting step of liposomepreparation, depending upon the site targeted for liposome delivery. Forexample, lactosyl ceramide, and peptides that target the LDL receptorrelated proteins, such as apolipoprotein E3 (“Apo E”) has been useful intargeting the liposomes of the present invention to the liver. Inparticular, the use of the Apo E ligand resulted in a 4-fold greatergene expression in the liver.

[0066] In addition, magnetic resonance imaging shows that the half-lifeof sandwich liposome complexes is at least 5 hours in circulation. Thisdemonstrates that the sandwich liposomes have time to reach the intendedtarget intact.

[0067] Alternatively, monoclonal antibody fragments may be used totarget delivery to specific organs in the animal including brain, heart,lungs or liver.

[0068] The present method to add ligands only to the outer surface aftercomplex formation is unique and has the advantage of avoiding disruptionof the biologically active agent liposome complex formation due tosteric or ionic interactions with the targeting ligand.

[0069] One embodiment of the liposomes of the present invention arecompletely invaginated with two concentric lamellae and a small orificewith an approximate diameter of 50 nm (FIG. 3A), which are called a“vase structure” (FIG. 3C). For example, of 536 vesicles observed, 88%were invaginated structures, and only 12% were the typical smallunilamellar vesicles (SUVs). The invaginated sandwich liposomes of thepresent invention include other structures, such as, bilamellar vases,bilamellar and unilamellar tubular shapes, unilamellar erythrocyteshapes, and unilamellar bean shapes.

[0070] Liposomes are examined after each step in the process of makingthe sandwich liposomes and unilamellar spheres are observed untilextrusion through the 0.1 μm filter. This extrusion step producesinvaginated structures with excess surface area. Performing only mildsonication (see Example 1) prior to extrusion is also recommendedbecause high frequency sonication of DOTAP:Chol liposomes produces onlySUVs and micelles. The bulk (88%) of sandwich liposomes prepared by theinstant method are bilamellar and unilamellar invaginated vesicles.

[0071] It appears that the biologically active agent adsorbs onto theinvaginated and tubular liposomes via electrostatic interactions (FIG.4). Attraction of a second liposome to this complex results in furthercharge neutralization. If the liposomes are of unequal size, expandingelectrostatic interactions with the biologically active agent causeinversion of the larger liposome and total engulfment of thebiologically active agent. These structures are sandwich liposomes.Inversion can occur in these liposomes because of their excess surfacearea, which allows them to accommodate the stress created by the lipidinteractions with the biologically active agent.

[0072] For example, DNA binding reduces the surface area of the outerleaflet of the bilayer and induces the negative curvature due to lipidordering and reduction of charge repulsion between cationic lipidheadgroups. Condensation of the internalized lipid sandwich expands thespace between the bilayers and may induce membrane fusion to generatethe apparently closed structures in FIGS. 3B, D. Interaction of morethan two liposomes may create the more complex structures seen in thismicrograph. The predicted thickness of 10.5 nm for DNA sandwichedbetween 2 bilayers (FIGS. 3D, G) is in agreement with observedmeasurements of these areas as shown in FIG. 3B. In addition, thethickness of DNA+lipid is confirmed by small angle x-ray scatteringanalyses (FIG. 8).

[0073] Alloying soft bilayers that contain dioleoyl chains withcholesterol is known to increase the stretching elastic modulus by up toan order of magnitude (Lasic and Needham (1995)). Liposomes that havemechanically weaker bilayers cannot efficiently undergo an inversion,and the agent complexed to these liposomes is less protected in thecirculation. The presence of appropriate levels of cholesterol in thebilayer provides sufficient strength to liposomes for efficient “vase”formation. The size, mechanical strength, and flexibility of the lipidvesicles as well as the biologically active agent-liposome ratio arecritical for this self-assembly mechanism of DNA condensation on theinterior of invaginated liposomes. This model predicts that anapproximate ρ value of 2 will be preferred to neutralize all chargeassociated with the biologically active agent by generating the lipidbilayer “sandwich”. This also demonstrates that the outside of thesandwich liposome complexes will be positively charged and free ofbiologically active agent.

[0074] The present invention clearly shows that cholesterol is anefficient neutral lipid in a liposome complex for in vivo DNA delivery.High content of cholesterol is known to increase the stability ofliposomes. The presence of cholesterol stabilizes bilayers and complexesin the plasma against mechanical breakage upon adsorption of plasmacomponents. In addition, DOTAP is an effective cationic lipid. Thecombination of DOTAP:Chol to produce cationic liposomes under thespecific method of the present invention resulted in liposomes withunique and useful properties for in vivo gene delivery. The presentinvention provides an up to 150-fold improvement in gene expression inseveral organs using extruded DOTAP:Chol liposomes for systemic DNAdelivery as compared with prior art techniques. This vast improvementallows increased efficiency of gene transfer in vivo.

[0075] Unlike other cationic liposomes, DOTAP:Chol liposomes, in oneembodiment, form stable DNA complexes over a broad range of DNA:liposomeratios. This flexibility allows optimization of the complexes for invivo delivery to different tissues. Most tissues other than lung arevery sensitive to this ratio (corresponding to ρ=2); therefore, thisratio must be carefully optimized for each DNA concentration. Thestability of DOTAP:Chol at high concentrations of liposome and DNAallows for increased concentrations of DNA to be delivered andexpressed. An effective liposome system must protect DNA in thecirculation, yet be able to deliver the DNA effectively to tissues.These properties have been achieved with the DOTAP:Chol liposomes as aresult of their more cohesive bilayer and their ability to internalizeand therefore protect DNA.

[0076] The following examples serve to illustrate further the presentinvention and are not to be construed as limiting its scope in any way.

[0077] While the invention is described above in relation to certainspecific embodiments, it will be understood that many variations arepossible, and that alternative materials and reagents can be usedwithout departing from the invention. In some cases such variations andsubstitutions may require some experimentation, but will only involveroutine testing.

[0078] The foregoing description of the specific embodiments will sofully reveal the general nature of the invention and others can, byapplying current knowledge, readily modify and/or adopt for variousapplications such specific embodiments without departing from thegeneric concept, and therefore such adaptations and modifications areintended to be comprehended within the meaning and range of equivalentsof the disclosed embodiments.

[0079] All of the references mentioned in the present application areincorporated in toto into this application by reference thereto.

EXAMPLE 1

[0080] DOTAP:Chol liposomes, as well as other liposome formulations,were prepared using the following procedure: the cationic lipid (DOTAPor DDAB) was mixed with the neutral lipid (Chol or DOPE) at equimolarconcentrations. The mixed powdered lipids were dissolved in HPLC-gradechloroform (Mallinckrodt) in a 1 L round bottomed flask. The clearsolution was placed on a Buchi rotary evaporator at 30° C. for 30 min tomake a thin film. The flask containing the thin lipid film was driedunder vacuum for 15 min. The film was hydrated in 5% dextrose in water(“D5W”) to give a final concentration of 20 mM DOTAP (or 20 mM DDAB) and20 mM Chol (or 20 mM DOPE), and is referred to as 20 mM DOTAP:Chol. Thehydrated lipid film was rotated in a 50° C. H₂O bath for 45 min and thenat 35° C. for an additional 10 min. The mixture was allowed to stand inthe parafilm-covered flask at room temperature overnight. On thefollowing day, the mixture in the flask was sonicated for 5 min at 50°C., transferred to a tube, and was heated for 10 min at 50° C. Themixture was sequentially extruded through decreasing size filters: 1 μm,0.45 μm, 0.2 μm, and 0.1 μm (Whatman) using syringes. Whatman Anotopfilters, 0.2 μm and 0.1 μm, were used. Portions of the liposome mixturethat did not pass through the first 0.1 μm filter, were heated again at50° C. for 5 min before passing through a new 0.1 μm filter. Filteredfractions were pooled and stored under argon gas at 4° C. DOTAP and DOPEwere purchased from Avanti Polar Lipids. DDAB was purchased from SigmaChemical Company, and highly purified cholesterol was purchased fromCalbiochem.

EXAMPLE 2 DNA-sandwich Liposomes

[0081] DNA:liposome complexes were prepared the day prior to their usein an animal host. DNA was diluted in D5W (5% dextrose in water), andstock liposomes were diluted in D5W to produce various ratios ofDNA:liposomes. The final volumes of both the DNA solution and theliposome solution used for mixing were equal. Dilutions and mixings wereperformed in 1.5 ml Eppendorf tubes with all reagents at roomtemperature. The DNA solution was added rapidly at the surface of theliposome solution using a pipette tip. The DNA:liposome mixture wasmixed rapidly up and down twice using a Pipetman. DNA:liposome complexeswere stored overnight at 4° C.

[0082] The protocol used for DNA preparation, referred to herein as theDebs protocol, is a variation of the alkaline lysis procedure describedby Maniatis (Sambrook et al. (1989)), and includes a 2 hour Proteinase Kdigestion step immediately following RNase A digestion. This methodconsistently produced about 20-fold greater amounts of DNA than thealkaline lysis procedure followed by polyethylene glycol (PEG)precipitation, and yields were in the range of 10 to 28 mg of plasmidDNA per liter of bacterial culture. This DNA preparation protocolresulted in no toxicity of DNA:liposome complexes in mice. Endotoxinlevels of DNA prepared by the different methods were determined usingthe chromogenic limulus amebocyte lysate assay (Kinetic-QCL;BioWhittaker). Endotoxin levels of 120 Endotoxin Units (EU)/μg DNA, 16EU/μg DNA, and 8 EU/μg DNA were determined for the Maniatis alkalinelysis method followed by PEG precipitation, the Qiagen Maxi-prep Kit,and the Debs protocol for preparation of plasmid DNAs, respectively. Nogenomic DNA, small DNA fragments, or RNA were detected in the DNAprepared by the Debs protocol, and the OD_(260/280) ratios of allplasmid DNA preparation were 2.0.

EXAMPLE 3 In Vivo Administration of DNA-sandwich Liposomes

[0083] For in vivo intravenous administration, 6-weeks old (˜20 g)BALB/c mice were injected in the tail vein with 200 μl of DNA:liposomecomplexes using a 27-gauge syringe needle. Samples were placed at roomtemperature for 1 hour prior to tail vein injection. Mice weresacrificed 24 hours post-injection, and the organs were harvested andquickly frozen on liquid nitrogen. Tissue extracts were prepared aspreviously described (Stribling et al. (1992)). Lymph node extractsconsisted of pooled mesenteric, axillary, iliac, submandibular, andinguinal lymph nodes. ELISAs were performed using the BoehringerMannheim CAT ELISA kit. All CAT protein determinations were correctedfor any CAT immunoreactivity detected in control tissues, and the lowestlevels of CAT protein reported for any experimental tissues were atleast 3-fold higher than background. Protein determinations wereperformed using the Micro BCA kit (Pierce). This work was conducted inaccordance with NCI/FCRDC guidelines using an approved animal protocol.

[0084] In order to demonstrate that the DNA is protected within theliposome, DNA-sandwich liposomes can be subjected to filtration throughpolysulfone filters of various pore sizes. The liposomes maintain fullactivity through the 1.0 μm and 0.45 μm filters, demonstrating that theDNA is fully sequestered and protected from the exterior. (See Table 1)If the DNA had been attached to the outside of the liposome, its proteinexpression activity would have been lost by filtering, since polysulfonestrips DNA from complexes carrying it on the outside.

[0085] Table 1 also illustrates that the DNA-sandwich liposomes capableof efficient DNA delivery are preferably larger than 200 nm in size.Particularly preferred DNA-sandwich liposomes include those between 200nm and 450 nm in size. The exact size may vary with the DNA containedtherein, the route of administration and the condition being treated.The skilled artisan can readily determine the appropriate size complexesbased upon these parameters. TABLE 1 CAT production in organs using sizefractionated DNA: liposome complexes Total CAT protein produced (ng)* No1.0 μm 0.45 μm 0.2 μm Organ Filtration Filtration Filtration FiltrationLung 168.6 207.0 154.8 68.4 Heart 8.5 7.6 6.6 1.8 Liver 5.5 4.6 6.0 2.4Muscle (quadricep) 5.0 6.0 5.7 1.5 Kidney 1.0 1.6 1.2 0.6 Thymus 0.9 0.81.2 0.1 Colon 0.4 0.7 0.9 0.6 Spleen 0.2 0.2 0.3 0.1 Lymph nodes 0.1 0.10.1 0.03 Brain 0.1 0.1 0.2 0.1

EXAMPLE 4 Comparison of Liposome Combinations

[0086] In order to demonstrate the unexpected nature of the presentinvention, the capacity of different liposome formulations to formcomplexes with DNA was examined. Various concentrations of liposomeswere mixed with DNA in a final volume of 200 μl, and the absorbance at400 nm was determined for 1:20 dilutions of each sample. TheDNA:liposome phase diagram showed that DOTAP containing liposomes stablycomplexed large amounts of DNA over a wide range of DNA:liposome ratios(FIG. 1A). Soluble complexes using DDAB liposomes had lower maximal DNAconcentrations and were stable over a narrow range using either DOPE orChol. A maximum of 82.5 μg of DNA was optimally complexed with 5 mM DDABformulations in a 200 μl final volume without causing precipitation.

[0087] On the basis of their physicochemical properties, these liposomeformulations were complexed to DNA and introduced into mice by tail veininjection. Systemic DNA delivery and gene expression were compared using1:1 formulations of DDAB:Chol, DDAB:DOPE, DOTAP:Chol, and DOTAP:DOPEliposomes, each at 5 mM, complexed to 82.5 μg plasmid DNA in a 200 μlfinal volume. This DNA:liposome ratio was found to be optimal for invivo expression of DNA delivered by DDAB:Chol liposomes (Liu et al.(1995)). At 24 hours postinjection the mice were sacrificed, and thelevels of CAT production in the lung were determined (FIG. 1B). Bothextruded and non-extruded preparations of the above lipid formulationswere injected into mice and the results compared to each other and tothose obtained by other investigators. The non-extruded liposomepreparations resulted in lower expression in all cases compared to thecorresponding extruded preparations. CAT production using DDAB:Cholliposomes prepared by sonication without extrusion, was in excellentagreement with CAT production reported using the same conditions (Liu etal. (1995)), based on the specific activity of CAT at 100,000 U per mgper min.) and the identical plasmid (The CAT plasmid used in allexperiments were p4119 (Liu et al. (1995)). Using additional heating andextrusion steps (see Example 1) in the preparation of DDAB:Cholliposomes prior to mixing with DNA, increased expression 2-fold.Interestingly, the extruded DOTAP:Chol-DNA liposome complexes produced50-fold greater amounts of protein expression in the lung compared tothe highest levels that have been reported using sonicated DDAB:Chol inthe same tissue (Liu et al. (1995)). In addition, the level of CATprotein produced in the lung using this novel formulation was greaterthan 50-fold compared to that using sonicated DOTMA:DOPE (DOTMA isN-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (alsoknown as Lipofectin)) (Zhu et al. (1993)) or sonicated DOTIM:Chol (DOTIMis1-[2-(9(Z)-Octadecenoyloxy)ethyl]-2-(8(Z)-heptadecnyl)-3-(2-hydroxyethyl)imidazoliniumchloride) (Solodin et al. (1995)). Extruded DOTAP:DOPE-DNA liposomecomplexes and extruded DDAB:DOPE-DNA liposome complexes produced farless CAT in the lungs compared to DOTAP:Chol-DNA liposome complexes.

[0088] To determine whether other extruded DOTAP:DOPE formulations wouldimprove delivery by using the optimal DNA:liposome ratio for the lungs,4 mM DOTAP:DOPE complexed to 100 μg of DNA were injected into mice. CATproduction remained 50-fold lower than that observed with 4 mMDOTAP:Chol-DNA liposome complexes (FIG. 2E). In addition, CAT productionwas determined using other extruded liposome formulations including 4 mMDOTAP-DNA liposome complexes, 4 mM DOTAP:Chol: DioleoylPhosphatidylserine (DOPS)-DNA liposome complexes, and 4 mM DOTAP:OleoylPhosphatidylcholine (DOPC)-DNA liposome complexes. As shown in FIG. 2E,DOTAP and all other formulations were not as effective as DOTAP:Chol forgene expression in vivo, and these formulations did not provide as broada biodistribution as that using DOTAP:Chol (data not shown). Addition ofsmall amounts of DOPS, 5%, to DOTAP:Chol (50:45) dramatically reduced invivo gene expression (FIG. 2E), and use of DOPC instead of Choleliminated in vivo gene expression almost entirely (FIG. 2E). Both DOPSand DOPC contain large headgroups and could perhaps interfere with theDNA:liposome assembly shown in our model (FIG. 4).

EXAMPLE 5

[0089] In order to evaluate in vivo expression, 100 μg of DNA were mixedwith varying concentrations of DOTAP:Chol. The highest levels of CATwere produced in lungs using 3 mM and 4 mM DOTAP:Chol complexed to 100μg DNA, with an approximate ± charge ratio ρ=2. However, significantexpression was obtained at all ratios (FIG. 2A). Furthermore, themaximal expression using 100 μg DNA was greater in lungs than thatproduced from injections of 5 mM DOTAP:Chol complexed to 150 μg DNA, andthis expression was approximately 100-fold greater than previouslyreported using other cationic liposomes (Zhu et al. (1993); Philip etal. (1993); Solodin et al. (1995); Liu et al. (1995) based on thespecific activity of CAT at 100,000 U per mg per min.). In addition,complexes of 3 mM to 4 mM DOTAP:Chol mixed with 100 μg DNA were stableupon storage at 4° C. for longer periods than complexes mixed with 125μg and 150 μg DNA at optimal DNA:liposome ratios. To determine whetherDOTAP:DOPE formulations would improve delivery by using the optimalDNA:liposome ratio, 3 mM DOTAP:DOPE complexed to 100 μg DNA wereinjected. However, the levels of CAT produced remained 50-fold lowerthan that produced by 3 mM DOTAP:Chol-DNA liposome complexes.

EXAMPLE 6

[0090] Using different concentrations of DOTAP:Chol complexed to 50 μgof DNA, there were more dramatic differences in the dose-response in thelung as compared to formulations containing higher amounts of DNA.Optimal production of CAT in the lung was observed after injection ofcomplexes containing 2 mM DOTAP:Chol complexed to 50 μg of DNA (FIG.2A). Thus, optimal expression at all DNA concentrations was achieved atcomparable DNA:liposome ratios namely approximate ± charge ratio ρ=2.However, the lower colloidal concentration of DNA and liposomes using 50μg DNA reduced the expression levels even at the optimal DNA:liposomeratio. In addition, CAT production fell sharply at DNA:liposome ratioseither lower or higher than the optimum.

[0091] To assess the amount of protein expressed in each tissue, totalprotein production was calculated for animals receiving 3 mM or 4 mMDOTAP:Chol complexed to 100 μg DNA (Table 2). Data showed that highlevels of gene expression could be obtained using DOTAP:Chol as asystemic delivery vehicle. Although expression in lung was relativelyinsensitive to the ratio of DNA:liposome, most other tissues showed a 2to 4-fold decrease in protein production using 4 mM instead of 3 mMDOTAP:Chol. Thus, the greater sensitivity to DNA:liposome ratio notedfor heart in FIG. 2C may apply to most tissues other than the lung.TABLE 2 Effect of DNA: lipid ratio on organ distribution of proteinproduction. Total CAT Protein produced (ng)* 4 mM 3 mM DOTAP: DOTAP:Organ Chol Chol Lung 170.4 166.8 Heart 8.7 3.5 Liver 5.6 3.4 Muscle(quadricep) 5.1 1.8 Kidney 0.9 0.6 Colon 0.4 0.1 Spleen 0.3 0.1 LymphNodes 0.2 0.1 Thymus 0.2 0.1 Brain 0.2 0.1

EXAMPLE 7

[0092] Because the in vitro data showed that DOTAP:Chol formed stablecolloidal complexes over a wide range of DNA:liposome ratios (FIG. 1A),different groups of mice were injected with complexes consisting of 0.5mM to 6 mM DOTAP:Chol mixed with 50 to 175 μg DNA in a 200 μl finalvolume. The goal was to determine the optimal DNA:liposome ratio for theDOTAP:Chol system. Results from initial experiments showed that DNAdelivery and gene expression were further increased by optimizing thisDNA:liposome ratio. Maximal CAT production in mouse lung was produced bythe use of 3-4 mM DOTAP:Chol complexed to 100 μg DNA (corresponding to a± charge ratio {ρ} of 2, FIG. 2A). The highest level of CAT productionwas approximately 100-fold greater than previously achieved (Zhu et al.(1993); Philip et al. (1993); Solodin et al. (1995); Liu et al. (1995)).The CAT activity was based on the specific activity of CAT at 100,000 Uper mg per min. Although higher amounts of DNA formed stable complexeswith these liposomes, toxicity was induced after 200 μl injections of250 and 300 μg DNA complexed with 9 mM and 10 mM DOTAP:Chol,respectively.

EXAMPLE 8 Tissue Expression Levels

[0093] Using the optimal DNA:liposome ratio and colloidal concentrationfor the DOTAP:Chol system identified in these experiments, (3 mMDOTAP:Chol complexed to 100 μg DNA), CAT production in other tissues wasstudied (FIG. 2B). Significant amounts of CAT were produced in alltissues examined (FIG. 2B). Approximately 75 to 150-fold greater amountsof CAT were produced in the lung, heart, liver, muscle, and kidney thanpreviously reported using other cationic lipids (Zhu et al. (1993);Philip et al. (1993); Solodin et al. (1995); Liu et al. (1995)) based onthe specific activity of CAT at 100,000 U per mg per min. Expression inthe heart was optimal at 3 mM DOTAP:Chol+100 μg DNA and decreasedmarkedly at other ratios (FIG. 2C). For lymph nodes and spleen, thelevels of CAT protein produced were about 25-fold and 2-fold greaterthan prior reports, respectively. CAT production in all tissues wasdetermined for every formulation. It was found that CAT production wasoptimal using 3 mM DOTAP:Chol complexed to 100 μg DNA in all tissuesexcept for lymph nodes. Gene expression in lymph nodes was optimal using5 mM DOTAP:Chol complexed to 150 μg DNA, and CAT production wasincreased 75-fold over that previously reported. Quantitation of CATproduction has not been previously reported in the thymus, skin, tail,colon, and brain, although expression has been noted in some of thesetissues (Zhu et al. (1993); Philip et al. (1993); Solodin et al. (1995);Liu et al. (1995); Thierry et al. (1995)).

[0094] If nucleated cells in the blood were transfected by thesystemically delivered complexes, expression of CAT in some tissues mayresult from blood included in the specimens. This was not the case inthe lung, because CAT production in whole blood, 47 pg/mg total protein,was much lower than in the lungs obtained from the same mice (FIG. 2D).Furthermore, CAT concentration measured in the lungs of exsanguinatedmice was elevated about 30%, because the total protein levels were lowerin the absence of blood. CAT levels for most other tissues from theexsanguinated mice showed either increased concentrations of CAT orconcentrations similar to nonbled mouse tissues. CAT concentrationlevels decreased in the tail, skin, and brain for samples assayed fromexsanguinated mice, suggesting that CAT production detected in thesetissues may, in part, be contributed by nucleated blood cells.

EXAMPLE 9 Targeting Ligands

[0095] Succinylated asialofetuin was made as previously described (Kaneoet al. (1991)), and 4 mM DOTAP:Chol was complexed with 100 μg of DNA ina 200 μl final volume. These complexes were filtered through a 0.45 μmpolysulfone filter (Whatman). Succinylated asialofetuin, to yield afinal concentration of 0.2 mg/ml, was added to these filteredDNA:liposome complexes using a Pipetman pipet tip. This mixture wasmixed slowly up and down twice in the pipet tip. DNA:liposome complexeswere stored overnight at 4° C. No precipitation of theDNA:liposome-asialofetuin complexes occurred. These complexes wereinjected into the tail vein of mice as described above.

[0096] Succinylated asialofetuin is highly negatively-charged;therefore, it can bind tightly to the positively charged surface ofliposomes that contain DNA between the invaginated liposomes. There wasa dose-dependent rise in the OD₄₀₀ after addition of succinylatedasialofetuin. The absorbance at 400 nm was determined for 1:20 dilutionsof the following DNA:liposome complexes with or without succinylatedasialofetuin. DOTAP:Chol-DNA liposome complexes had an OD₄₀₀ of 0.815;and DOTAP:Chol-DNA+0.1 g/ml succinylated asialofetuin andDOTAP:Chol-DNA+0.2 mg/ml succinylated asialofetuin had OD₄₀₀ readings of0.844 and 0.873, respectively. Addition of succinylated asialofetuin atamounts greater than 0.2 mg/ml produced immediate precipitation ofDNA:liposome complexes. These observations show that succinylatedasialofetuin strongly interacts with the DOTAP:Chol-DNA liposomecomplexes. Succinylated asialofetuin was added to the surface of theliposome complexes to achieve greater gene expression in the liver.Asialofetuin is an asialoglycoprotein containing terminal galactosylresidues and has been used to efficiently target liposomes to the liver.(Spanjer and Scherphof (1983); Spanjer et al. (1984); Dragsten et al.(1987); Murahashi and Sasaki (1996); Hara et al. (1995)). In addition,changing the surface charge on the outside of the DNA:liposome complexesto reduce ionic interactions with endothelial proteoglycans (Mislick andBaldeschwieler (1996)) may also facilitate organ-specific delivery.

[0097] Addition of succinylated asialofetuin to preformed DNA:liposomecomplexes provided a ligand for the hepatic asialoglycoprotein receptor(Ashwell and Harford (1982)) and increased CAT production in the liverseven-fold (FIG. 7). This targeting was specific for the liver, as CATexpression in other organs shown in FIG. 2B was not increased. Thiswidely used ligand was employed solely to demonstrate feasibility of thepresent method for adding ligands.

EXAMPLE 10 Cyro-Electron Microscopy

[0098] Thin films were prepared by dipping and withdrawing a 700-meshcopper grid (3 mm diameter, 3 to 4 μm thick) in the liposome or theDNA:liposome suspensions. After excess liquid was removed by blotting,the thin films that formed between the bars of the grid were vitrifiedin melting ethane. After cryotransfer, the specimen was observed at−170° C. in a Philips CM 12 microscope at low dose, 120 kV 19.

[0099] To examine the mechanism of the high in vivo gene delivery, theDNA-sandwich:liposome complexes were examined by studying theirmorphology as well as that of non-DNA associated liposomes withcryo-electron microscopy. Extruded DOTAP:Chol liposomes in the absenceof DNA showed many completely invaginated liposomes with two concentriclamellae and a small orifice. Most liposomes were spherical with anapproximate diameter of 50 nm.

[0100] Vase structures represented one-third of the entire liposomepopulation. Large tubular structures were also observed, explaining thesomewhat larger size of approximately 240 nm, determined by dynamiclight scattering using a Coulter N4 particle size analyzer.

[0101] When these liposomes were complexed with DNA at optimalconcentrations, with a ± charge ratio ρ=2 (Lasic et al. (1997)), the DNAwas localized to the interior of the liposomes (FIG. 3B). DOTAP:DOPE(dioleoyl phosphatidyl-ethanolamine) and DOTAP:Chol complexes with DNAwere turbid colloidal solutions with mean particle size of 445 nm and405 nm, respectively. Particle size did not depend on dilution, andturbidity obeyed Beer Lambert Law indicating stability of thesecomplexes in vitro. DOTAP:DOPE liposomes also form “vase structures”;however, the orifices were larger and many spheres were formed (FIG.3E). In addition, there were many structures with little or no DNAassembled in the extruded DOTAP:DOPE liposomes (FIG. 3F), and the DNAwas frequently found on the outside of these liposomes (FIG. 3F).DDAB:DOPE and DDAB:Chol liposomes did not form “vase structures”. Theinternalization of DNA within “vases” is a unique feature of extrudedDOTAP liposomes and have not been observed for any other DNA:liposomecomplex studied by cryo-electron microscopy (Frederik et al. (1991)).The “vase structures” observed for DOTAP:Chol may contribute to the highsystemic delivery and gene expression achieved with these formulations.

Small Angle X-ray Scattering (SAXS)

[0102] DOTAP: Chol-DNA complexes were concentrated into a highly orderedstructure by centrifugation or drying, and both techniques produced thesame SAXS results. SAXS experiments were performed using a rotatinganode x-ray source GX-13 (Elliot, England) focused by two bent x-raymirrors. The 2-dimensional x-ray pictures were taken with a Franck-typecamera at 15 cm distance from the sample, using Kodak phosphorousscreens (Kodak, N.Y.) that were scanned by an image plate reader(PhosphorImager SI, Molecular Dynamics, Calif.). The radial intensityaverages were determined using our modification of the NIH-Image 1.57image processing program (Wayne Rasband, National Institutes of Health,MD). The results are shown in FIG. 8.

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We claim:
 1. A liposome composition comprising DOTAP and at least onecholesterol or cholesterol derivative.
 2. The liposome compositionaccording to claim 1, further comprising a biologically-active agent,thereby forming a sandwich liposome.
 3. The sandwich liposomecomposition according to claim 2 wherein the composition has a ρ valueequal to
 2. 4. The liposome composition according to claim 2, whereinthe biologically-active agent is a nucleic acid.
 5. The liposomecomposition according to claim 4 further comprising, adding a targetingligand thereby decorating exterior surface of said sandwich liposomewith the ligand.
 6. A DNA-sandwich liposome composition comprising astructure having lipid bilayers and DNA molecules positioned between twoor more sandwich liposomes, wherein ρ=2 and a size of 200-450 nm.
 7. ADNA-sandwich liposome comprising DNA, DOTAP and at least one of acholesterol or cholesterol derivative.
 8. The DNA-sandwich liposome ofclaim 7 further comprising one or more targeting ligands.
 9. A liposomeproduced by the steps comprising: i) heating DOTAP and at least onecholesterol or cholesterol derivative forming heated lipid components;ii) sonicating said heated lipid components; and iii) extruding lipidcomponents sequentially through filters of decreasing pore size.
 10. Theliposome of claim 9 further comprising a sandwich liposome, produced byadding a biologically-active agent to the liposomes.
 11. The liposome ofclaim 10 wherein the biologically active agent is DNA, thereby forming aDNA sandwich liposome.
 12. The liposome according to claim 11 furthercomprising, adding a targeting ligand thereby decorating the exteriorsurface of said DNA-sandwich liposome with the ligand.
 13. The liposomeaccording to claim 11 further comprising a second biologically activeagent.
 14. The lipesome of claim 11 wherein the DNA, DOTAP and at leastone cholesterol or cholesterol derivative carry a ρ value of
 2. 15. Amethod for preparing invaginated liposomes comprising the steps of: i)heating a mixture of DOTAP and at least one of cholesterol orcholesterol derivative forming heated lipid component; ii) sonicating said heated lipid components; and iii) extruding lipid componentssequentially through filters of decreasing pore size forming invaginatedliposomes.
 16. The method of claim 15, further comprising adding DNA tosaid invaginated liposomes forming DNA-sandwich liposomes.